FEMS Microbiology Ecology 51 (2005) 353–361
www.fems-microbiology.org
Diel changes in bacteriochlorophyll a concentration suggest rapid
bacterioplankton cycling in the Baltic Sea
Michal Koblı́z̆ek
a
a,*
, Joanna Stoń-Egiert b, Sławomir Sagan b, Zbigniew S. Kolber
c
Institute of Microbiology and Institute of Landscape Ecology CAS, Opatovický mlýn, 379 81 Tr̆ebon̆, Czech Republic
b
Institute of Oceanology PAN, Powstańców Warszawy 55, 81-712 Sopot, Poland
c
Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, CA 93901, USA
Received 31 May 2004; received in revised form 5 August 2004; accepted 24 September 2004
First published online 2 November 2004
Abstract
Aerobic anoxygenic phototrophs were recently found to constitute a significant portion of the marine microbial community.
These bacteria use bacteriochlorophyll-containing reaction centers to perform photoheterotrophic metabolism. A new instrument
for routine measurements of both chlorophyll a and bacteriochlorophyll a was used for monitoring anoxygenic phototrophs in
the Baltic Sea in late summer 2003. Bacteriochlorophyll a concentration ranged from 8 to 50 ng l1, with an average bacteriochlorophyll/chlorophyll ratio of 4.2 · 103. Moreover, diel trends in bacteriochlorophyll a signals were observed, with a distinct
decline occurring during daylight hours. Based on laboratory measurements this phenomenon was ascribed to the complete inhibition of bacteriochlorophyll synthesis by light, which, in combination with a concurrent turnover of the cells, resulted in a pigment decline. Following this explanation, we postulate that bacteriochlorophyll a can serve as a natural Ôpulse-and-chaseÕ
marker, allowing estimation of the mortality rates of anoxygenic phototrophs from the rates of pigment decline. Based on this
assumption, we suggest that the Baltic photoheterotrophic community was characterized by high turnover rates, in a range of
0.7–2 d1.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Aerobic anoxygenic photoheterotrophs; Aerobic photosynthetic bacteria; Bacteriochlorophyll a; Bacterioplankton turnover; Bacterial
mortality; Diurnal cycles; Photoheterotrophy
1. Introduction
Oxygenic photosynthetic organisms and heterotrophs are generally thought to represent the two major players in the marine carbon cycle. Chlorophyll
(Chl a) containing eukaryotic and prokaryotic phytoplankton utilize light as the main source of energy
Abbreviations: D, mortality or dilution rate; t, time; l, growth rate;
s1/2, half-life.
*
Corresponding author. Fax: +420 384 721 246.
E-mail address: [email protected] (M. Koblı́z̆ek).
for converting inorganic CO2 into organic molecules.
Heterotrophic organisms, on the other hand, consume
fixed organic carbon for growth, respiring it back to
CO2. Besides these two major trophic pathways, there
also exists third kind of metabolism – photoheterotrophy [1]. Photoheterotrophic organisms depend on a
supply of organic substrates for growth, but they also
utilize light energy to substitute a significant portion
of their respiratory requirements. These organisms,
called aerobic anoxygenic phototrophs (AAPs), account for a significant fraction of the marine microbial
community [2,3]. AAPs are strict aerobes, containing bacterial photosynthetic centers composed of
0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2004.09.016
354
M. Koblı́z̆ek et al. / FEMS Microbiology Ecology 51 (2005) 353–361
bacteriochlorophyll a (BChla). Interestingly, BChla
synthesis is inhibited by light and, therefore, it is exclusively constrained to the periods of darkness [4].
Although functionality of the photosynthetic apparatus
was verified by fluorescence measurements and CO2
fixation assays, these bacteria are not capable of truly
autotrophic growth, as they require a supply of organic
carbon [4,5]. The ability to utilize light energy appears
to offer an ecological advantage serving as an auxiliary
source of ATP. However, AAPs distribution, diversity
and ecological role in the marine environment remain
unclear.
The Baltic is a brackish, rather heterogenous, enclosed shelf sea system [6]. The plankton dynamics of
the Baltic Sea are characterized by two seasonal
blooms. The spring bloom (March–May) is typically
dominated by dinophytes and/or diatoms (80–95% of
the total biomass) with a minority of chlorophytes,
cryptophytes and cyanobacteria. This bloom contributes about half of the annual primary production.
The autumn bloom (August–September) is dominated
by cyanobacteria along with dinophytes and chlorophytes as other main contributors [7,8]. Earlier field
studies suggested that Baltic primary production is
mostly limited by nitrogen availability, but the activity
of nitrogen fixing cyanobacteria in some periods of the
year results in phosphorus limitation [9]. The patterns
of control of the Baltic bacterioplankton community
appear to be more complex. In early spring the bacterial community was found to be predominantly controlled by nitrogen availability and nanoflagellate grazing
[10]. In some studies a stimulation of growth by phosphorus was observed in late spring, whereas in summer
a great stimulation was induced by the combined addition of nitrogen and phosphorus [9]. Up until now,
there has been no data on the presence of anoxygenic
photoheterotrophs in the Baltic Sea.
Kinetic fluorometry is a convenient way of monitoring the abundance and physiological status of marine
phytoplankton [11]. Infra red fast repetition rate
(IRFRR) kinetic fluorescence measurements provided
the first evidence of AAPs activity in the Pacific Ocean
[2,3]. Later, the IRFRR instrument was used to characterize the AAPs distribution in the Black Sea [12].
Unfortunately, AAPs characterization using IRFRR
instrument requires laborious pre-concentration of the
samples. To address this problem, a new sensitive kinetic fluorometer was developed. This instrument is
based on dual modulation technology [13,14] and allows the simultaneous measurement of both Chl a
(phytoplankton) and BChl a (AAPs) fluorescence kinetics at a 100 kHz sampling rate. This instrument was
used to detect spatial heterogeneity and dynamic
changes of AAPs distribution in the Baltic Sea during
a late summer of 2003 survey aboard the Polish R/V
Oceania.
2. Materials and methods
2.1. Fluorometry
The newly designed kinetic fluorometer employs a
dual modulation technology as described earlier
[13,14]. The instrument was assembled using the standard PSI fluorometer control unit (FL200/PS, Photon
Systems Instruments Ltd., Czechia) and custom made
optics. The optical part utilized one PSI flashing unit
(SN-LF 8052, PSI Ltd, Czechia) populated with 73 blue
light emitting diodes (NSPB500S, 470 nm, Nichia, Japan). Forty nine of the diodes were used to provide
short intense measuring pulses (10 ls) and the rest
served as an actinic light source operating in a continuous mode.
The instrument sample chamber was made of stainless steel containing a spherical compartment (60 ml)
coated with a Teflon reflective layer, with an inlet and
an outlet for sample injection and removal. The chamber had three ports: one port was used to introduce excitation light, the other two ports were used to interface
with Chl a and BChl a detectors. The excitation port
was protected by a broadband blue-green filter (Schott
BG 39 analogue). Chl a was measured by a standard
chlorophyll a detector (SN-SL 103, PSI Ltd., Czechia)
utilizing a silicon PIN photodiode (S3590-02, Hamamatsu, Japan) and protected by a red 665 nm long pass filter (Oriel 51330, USA) and a 700 nm interference filter
(700.0/70/75-, Intor Inc., USA). The BChl a detector utilized a large area avalanche photodiode module (SD
630-70-72-641, Advance Photonix Inc., USA) operating
at an internal gain of 300, protected by a glass infrared
830 nm long pass filter (Oriel 51352, USA). Both signals
were further amplified 300· and digitized with a 16-bit
AD converter. The control unit served to program the
measuring protocols, and to record the acquired signals.
To estimate phytoplankton and AAPs abundance by
kinetic fluorometry, fluorescence transients were stimulated using 200 ms long pulses of actinic light. The fluorescence induction was registered by a sequence of
measuring (probing) flashlets at 0.5 ms intervals. Then,
the actinic light was switched off and the fluorescence
relaxation was followed with 50 measuring flashes at 2
ms intervals. The fluorescence signal was averaged over
100 repetitions, with 2 s of dark periods. A small portion
of Chl a fluorescence was observed above 830 nm (1%
of the signal at 700 nm). To discriminate between the
BChl a and the Chl a contribution at this spectral range,
phytoplankton was selectively inhibited with the herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea;
Diuron). DCMU inhibits Photosystem II of oxygenic
phototrophs whereas the bacterial reaction centers were
unaffected [15]. In the presence of 105 M DCMU, the
part of the signal originating from the phytoplankton
rapidly rose, reaching the maximum within about 3
M. Koblı́z̆ek et al. / FEMS Microbiology Ecology 51 (2005) 353–361
30-min intervals. The dilution rate was set to 1 day1.
The culture was grown in 12:12 h light–dark cycle.
The illumination was provided by a bank of luminescent
tubes providing irradiance of 150 lmol quanta m2 s1.
1.2
off
Fluorescence [V]
> 830 nm signal
700 nm signal
355
BChl a
0.9
2.4. Analytical procedures
Chl a
0.6
on
0.3
0.1
1
10
100
1000
Time [ms]
Fig. 1. Protocol for discriminating between BChl a and Chl a signals.
The phytoplankton cells were selectively inhibited by the addition of
DCMU (Photosystem II inhibitor), which caused the rapid rise of the
Chl a signal within 3 ms (thin line). The BChl a signal (thick line) was
not affected by DCMU, rising slowly and reaching its maximum within
about 80 ms. After switching off the actinic light (arrow), the BChl a
signal declined within 100 ms whereas the Chl a signal did not relax.
The signals were recorded at station F4 (Gulf of Finland) on 28 August
2003.
ms. The signal originating from the bacterial reaction
centers was not affected, rising slowly and reaching a
maximum at about 80 ms (Fig. 1), allowing separation
of both signals. The instrument sensitivity was sufficient
to allow processing of natural water samples with a
detection limit of 2 ng BChl a L1 and 30 ng Chl a L1.
2.2. Optics
In situ measurements of light absorption a(k) and
attenuation c(k) were performed with an ac-9 meter
(WetLabs, USA) at wavelengths of 412, 440, 488, 510,
532, 555, 650, 676, and 715 nm. The instrument was factory calibrated in pure water, and routinely checked for
stability by air-readings. A temperature correction was
applied to the 715 nm absorption and attenuation channels according to the factory manual, and a scattering
correction was applied to the absorption channels [16].
Cyclostat samples were collected by centrifugation
and extracted in 100% methanol. The bacteriochlorophyll a content was determined by absorption measurement using the molar absorption coefficient e771 = 54.8
mM1 cm1 [17]. BChl a was also estimated in vivo from
IRFRR measurements as described earlier [3]. Cell biomass was estimated from the optical density measurements at 650 nm.
To perform the pigment analysis 1 L samples of seawater were filtered onto GF/F filters and the filters were
stored in liquid nitrogen. Pigments were extracted by
grinding and sonication (2 min, 20 kHz, Cole Parmer,
4710 Series) in 3 ml 90% acetone at 4 C in the dark
for 2 h, after which the extracts were centrifuged (20
min, 5 C, 2150g, Beckman, GS-6R), clarified and then
subjected to chromatographic analysis.
The pigment composition was analyzed by HPLC
using a modified procedure of Mantoura [18,19]. The
chromatographic system was composed of HP1050
pump, HP1046 fluorescence detector, HP1100 diode array detector, Rheodyne injector (100 ll loop) and the
LiChroCARTe LiChrosphere 100 RP-18e (dimension:
250 · 4 mm, particle size 5 lm, MERCK) analytical column. Pigments were separated by the binary solvent system was 80:20 (v/v) methanol:1 M ammonium acetate
(A) and 60:40 (v:v) methanol:acetone (B). The 10 min
linear gradient (A–B) was followed by 22 min isocratic
hold (100% B) with 0.8 ml flow rate. Finally, the solvent
was changed back to A to equilibrate the system prior
the next sample injection. Pigments were detected by
the absorption detector at 440 nm and in parallel by a
fluorescence detector (431 nm excitation, 660 nm emission) in order to confirm the presence of chloropigments
in the samples.
2.3. Cyclostat setup
3. Results
Erythrobacter sp. NAP1 strain [5] was grown in a
water-jacketed (23 C) glass vessel (volume 2.7 L).
The growth medium was composed of natural seawater
enriched with 3 · 103 M glucose, 102 M (NH4)2SO4,
3 · 105 M NaH2PO4, trace metal mix (105 M sodium
iron (III) ethylenediaminetetra-acetate, 4 · 108 M
CuSO4, 8 · 108 M ZnSO4, 4 · 108 M CoCl2,
9 · 107 M MnCl2, 3 · 108 M Na2MoO4) and vitamins
(2 · 109 M biotin, 3.7 · 1010 M B12). The cell suspension was thoroughly stirred and air bubbled. The growth
medium was pumped in and out of the vessel for 60 s in
The field measurements were performed during the
‘‘Biooptics Cruise’’ in the Baltic Sea in late summer of
2003 (Fig. 2). The cruise consisted of two transects;
the first one from Gdańsk to Helsinki (25–28 August
2003) and the second one from Helsinki to Gdańsk (30
August–5 September 2003). During this period the
hydrological conditions were typical for the summer season. Surface layer (0–40 m) temperatures ranged from
15 to 16 C in the Eastern Gotland Basin, reaching up
to 18.5 C in the Gdańsk Basin (see Table 1). Significant
differences in the thickness of the mixed surface layer
356
M. Koblı́z̆ek et al. / FEMS Microbiology Ecology 51 (2005) 353–361
17˚
23˚
20˚
values of 7.1–7.2 psu. Weather conditions varied from
partially cloudy (4 September 2003) to total overcast
(30 August 2003). Winds ranged from 5–7 m s1 (26 August, 30 August to 3 September 2003) to stormy conditions with winds of 16–20 m s1 (27 August 2003).
Phytoplankton composition was rather heterogenous.
Based on pigment analyses the population was composed of dinophytes, diatoms and cyanobacteria with
a small contribution of green algae without any clearly
dominant group.
The variable fluorescence components of Chl a and
BChl a signals measured during the cruise are shown
in Fig. 3. The upper panel shows Chl a variable fluorescence (FV) signals overlaid with absolute Chl a concentrations as determined by HPLC analysis. In spite of
high temporal and spatial variability, Chl a concentration was slightly higher in the Southern Baltic and in
the Gulf of Finland, where the influx of nutrients stimulated growth of phytoplankton. Lower Chl a concentrations were observed in the central Baltic (Eastern
Gotland Basin, Fig. 3(a)). Based on the HPLC analyses
the Chl a abundance ranged from 3 to 9 lg Chl a L1.
Similar patterns of Chl a distribution could be also deduced from the water absorption at 676 nm (Fig. 4).
The BChl a signals (Fig. 3(b)), on the other hand, displayed a much different pattern. A clear south to north
gradient, with the lowest numbers in the Southern Baltic
(Gdańsk Basin), increasing toward the Gulf of Finland
was observed. In absolute terms the BChl a concentrations in the Baltic ranged from 8 to 50 ng BChl a L1.
The BChl a/Chl a ratios (mol:mol) (Fig. 5) were calculated assuming molecular weight of Chl a Mr = 893.5
and 911.5 for BChla. The average BChl a/Chl a ratio
was 4.24 ± 0.33 · 103. BChl a showed a trend similar
26˚
Helsinki
60˚
60˚
F6
F7
F3
F8
F2
F1
Stockholm
F4
F5
Tallinn
58˚
58˚
PY15a
F9
PY15b
F10
Riga
P63c
56˚
56˚
P63a
P1
P104
0
Gdansk
50
100
150 km
P110c
54˚
17˚
23˚
20˚
54˚
26˚
Fig. 2. Map showing the cruise track and the sampling stations. The
open symbols show stations of the first leg of the cruise from Gdańsk
to Helsinki (25–28 August 2003). The closed symbols show the stations
of the second leg from Helsinki to Gdańsk (30 August to 5 September
2003). Station F3 was revisited also during the second leg of the cruise.
(depth of the thermocline) were observed between various regions: 40–45 m at Gdańsk Bay, 20–25 m at Northern Baltic Proper, and 18 m at Eastern Gotland Basin
stations. Salinity within the upper 60 m displayed typical
Table 1
List of stations
Station
Latitude N
0
Longitude E
Date
Temperature
0
P1
P63c
F1
F2
F3
F4
54
56
58
58
58
59
50.0
10.2 0
38.5 0
48.3 0
57.2 0
38.7 0
19
19
21
21
21
24
19.4
05.8 0
24.4 0
37.2 0
47.3 0
08.7 0
8/25
8/26
8/27
8/27
8/27
8/28
18.5
18.1
16.4
16.7
16.4
16.5
F5
F6
F7
F3
F8
PY15a
F9
PY15b
F10
P63a
P104
P110c
P110c
59
59
59
58
58
57
57
56
56
55
54
54
54
21.5 0
15.2 0
08.2 0
57.1 0
48.1 0
41.9 0
27.8 0
58.6 0
48.6 0
39.2 0
34.7 0
30.1 0
29.9 0
22
22
21
21
21
20
19
19
19
18
18
18
18
35.9 0
18.8 0
59.2 0
49.3 0
30.9 0
10.3 0
56.5 0
27.7 0
21.7 0
56 0
47.6 0
56.2 0
56.5 0
8/30
8/30
8/30
8/30
8/30
8/31
8/31
9/01
9/01
9/02
9/03
9/03
9/04
15.9
15.6
14.7
15.2
15.1
15.7
15.7
15.6
13.3
18.5
17.4
16.3
17.8
M. Koblı́z̆ek et al. / FEMS Microbiology Ecology 51 (2005) 353–361
10
1.5
6
1.0
4
FV Chl a [V]
-1
Chl a [µ
µg L ]
8
0.5
2
0
54
55
56
57
(a)
58
59
60
0.0
61
Latitude N
60
160
120
-1
40
30
80
20
FV BChl a [mV]
BChl a [ng L ]
50
40
10
0
54
55
56
57
(b)
58
59
60
0
61
Latitude N
Fig. 3. Summary of Chl a (upper panel) and BChl a (lower panel)
distribution in surface waters. The upper panel shows Chl a variable
fluorescence signal (empty squares) and absolute Chl a concentrations
as determined by HPLC (filled diamonds). The BChl a values were
estimated from variable fluorescence signal (lower panel). The empty
symbols represent the data acquired during the first leg of the cruise
from Gdańsk to Helsinki. The closed symbols represent data acquired
during the second leg from Helsinki back to Gdańsk. The dashed
trend-lines were obtained as quadratic fits of the respective data points.
357
to that of the distribution of colored dissolved organic
matter (CDOM) as estimated from water absorption
at 412 nm (Fig. 4). As it was suggested previously,
CDOM in the open-sea Baltic waters originates mostly
from decomposed phytoplankton [20]. This indicates
that AAPs distribution might have been controlled by
the availability of dissolved organic matter.
In addition to this dominant trend, another pattern in
BChl a abundance was pervasive. We have repeatedly
observed a gradual decline of the BChl a signal during
daylight hours (Fig. 6). The only exception to this trend
was observed on 27 August 2003, when the BChl a content remained almost constant. The daily decline in
BChl a appears to be unrelated to the spatial variability,
as it was also observed at stations occupied over a significant portion of a day. Neither can this decline be attributed to change in the BChl a fluorescence quantum
yield. This assumption is supported by the fact that
the anoxygenic photosynthetic bacterium Rhodobacter
capsulatus was shown to be insensitive to photoinhibition and to lacked any non-photochemical quenching
mechanisms [21]. Similarly, we have not observed any
non-photochemical quenching in our environmental isolates of AAPs, even at irradiances exceeding 1500
lmol photons m2 s1 (Koblı́z̆ek, unpublished). Instead,
the recorded decline in the fluorescence signals appears
to reflect true changes in the BChl a concentration.
We postulate that the daily decline in BChl a is
caused by the natural loss term of AAPs due to grazing
and/or viral infection. It is well established that AAPs
accumulate BChl a exclusively in the dark [4,22] since
the pigment synthesis is inhibited by light levels of just
a few lmol quanta m2 s1 [23,24]. During the daytime
the cells grow and divide, but BChl a which accumulated
8
A676
A412
-3
1.1
1.0
0.15
-1
A412 [m ]
-1
A676 [m ]
0.9
0.10
0.8
0.7
BChl a/Chl a ratio 10
0.20
6
4
2
0.05
0.6
0.00
54
55
56
57
58
59
0.5
60
Latitude N
Fig. 4. Absorption coefficients determined in surface waters along the
cruise transect. Water absorption at 412 nm is used as a proxy for the
presence of colored dissolved organic matter (CDOM). Absorption at
676 nm is a proxy for chlorophyll. The trend-lines were obtained as
quadratic fits of the respective data points.
0
54
55
56
57
58
59
60
Latitude N
Fig. 5. BChl a to Chl a ratios in the surface waters as measured along
the Baltic transect. Chl a values were determined by HPLC and the
BChl a was estimated from variable fluorescence signal. The dashed
trend-line was obtained as a linear fit of the obtained data. It shows the
relative increase of the BChl a content in the northern Baltic. The
average BChl a/Chl a ratio was 4.24 ± 0.33 · 103 (mol:mol).
358
M. Koblı́z̆ek et al. / FEMS Microbiology Ecology 51 (2005) 353–361
400
BChl a
Irradiance
BChl a [ng L-1]
50
300
40
200
30
PAR [Wm-2]
60
100
20
10
6
8
10
(a)
12
14
16
18
CET [hours]
20
400
BChl a
Irradiance
300
16
200
14
PAR [Wm-2]
18
BChl a [ng L-1]
0
20
der light-dark conditions, with the dilution rate, D, set
to 1 day1. The cell biomass, as estimated from optical
density measurements at 650 nm, was slightly rising
(Fig. 7). As the cyclostat was not in the steady state, this
increase can be described by the net growth rate constant lNET = 0.20 ± 0.01 day1. From the dilution rate
and lNET, we estimated the gross rate, lGROSS =
lNET + D, at 1.20 ± 0.01 day1. The BChl a concentration, however, displayed a clear diurnal cycle. During
the light period, the BChl a signal declined exponentially
(s1/2 16 h) and recovered in the dark (Fig. 7). This is
consistent with inhibition of BChl a synthesis in the light
[23]. In the dark the BChl a content started to rise only
2–3 h after the onset of the darkness. This indicates that
some enzymes of the BChl a biosynthetic pathway were
not available and had to be synthesized de novo. The
BChl a concentration then increased during the remaining dark period, until the onset of light in the morning
(Fig. 7).
Assuming no de novo BChl a synthesis, the BChl a
decline can be mathematically described as follows:
o½Bchl a=ot ¼ ½Bchl a D;
100
12
10
6
8
10
(b)
12
14
16
18
0
20
CET [hours]
½BChl at ¼ ½BChl at¼0 eDt ;
12
400
where [BChl a]t is BChl a concentration at time t. The
BChl a half-life s1/2 can be expressed as:
BChl a
Irradiance
s1=2 ¼ ln 2=D:
300
10
200
9
PAR [Wm-2]
11
BChl a [ng L-1]
where [BChl a] is the BChl a concentration, D is the dilution rate and t is time. Integrating this equation, a simple exponential relationship is obtained:
The observed decline in fluorescence signal was analyzed
by numerical curve-fitting using the single exponential
100
100
12
8
fluorescence
10
12
14
16
18
CET [hours]
Fig. 6. Diurnal decline of BChl a signal in the surface waters recorded
in the Northern Baltic Proper (30 August 2003, stations F5–F8, panel
a), the Eastern Gotland Basin (1 September 2003, station PY15b, panel
b), and the Gulf of Gdańsk (4 September 2003, station P110c, panel b).
The gray line shows the changes in photosynthetically active radiation
(PAR) during the day. The solid lines show the exponential decay fit of
the BChl a data. The details of the analyses are discussed in the text (a:
R2 = 0.901, b: R2 = 0.851, c: R2 = 0.915). CET = Central European
time.
during the previous night decays with a rate defined
exclusively by the loss term, resulting in the observed decline of BChl a.
To verify this hypothesis we performed laboratory
experiments with a continuous culture of AAPs. Erythrobacter sp. strain NAP1 [5] was grown in cyclostat un-
-1
8
10
BChl a
O.D. [m ]
6
(c)
0
20
-1
7
BChl a [µ
µg L ]
80
60
8
40
6
20
4
O.D.650 nm
0
2
6
12
18
24
30
36
Time [hours]
Fig. 7. Changes in BChl a content in a continuous culture of
Erythrobacter sp. NAP1 grown under 12:12 light dark conditions.
The dark periods are marked by black bars on the top of the figure.
The dilution rate was set to 1 day1 and the illumination was about
150 lmol quanta m2 s1. The dashed lines represent fitted single
exponential kinetics of the fluorescence data. The calculated rate
constant of the BChl a decay was 1.06 ± 0.03 day1. The optical
density rose with a rate of 0.20 ± 0.01 day1 (solid line), which gives
the growth rate of 1.20 ± 0.01 day1.
M. Koblı́z̆ek et al. / FEMS Microbiology Ecology 51 (2005) 353–361
3
-1
BChl a half-life
mortality rate
24
2
18
12
1
6
0
54
55
56
57
58
Mortality rate D [day ]
BChl a half-life [hours]
30
59
0
60
Latitude N
Fig. 8. BChl a half-lives and corresponding mortality rates as
determined from the analysis of the field data. Data points correspond
to (from left to right): Station P110C, the Gulf of Gdańsk, 4 September
2003; Station PY15b, the Eastern Gotland Basin, 2 September 2003
and transect F5–F8, the Northern Baltic Proper, 30 August 2003. The
trend-lines were obtained as quadratic fits of the BChl a decay or
AAPs mortality data, respectively.
decay kinetics (Fig. 7). The obtained rate constant of
1.06 ± 0.03% (r2 = 0.965) demonstrates that BChl a followed the theoretical wash-out kinetics of the cyclostat.
Moreover, this experiment demonstrated that the
changes of BChl a concentration in the light follow the
loss term (in this case the washing out the cells from
the cyclostat), independently of the actual growth-rate
of the culture. The washing out of cells from the cyclostat simulates mortality of bacterial population. Therefore, the same formalism can be applied to explain
and to analyze the observed BChl a decays to assess
the AAPsÕ loss term (mortality) in situ.
Following this approach we estimated the AAPs
mortalities D and the half-lives s1/2 in their natural
environment. The mortality rates displayed a clear
south-to-north trend (Fig. 8), similar to that of AAPs
distribution. The lowest mortality rates were observed
in the Gulf of Gdańsk where AAPs cycled with the rate
of 0.68 ± 0.09 day1 (s1/2 24 h). The mortality increased in the Central Baltic (D = 1.45 ± 0.34 day1,
s1/2 = 11.5 h) and it was the highest in the Northern Baltic Proper (D = 2.17 ± 0.38 day1, s1/2 = 7.7 h). The data
from the Gulf of Finland (28 September 2003) suggest
even higher mortality rate, however, the small number
of measurements does not allow rigorous mathematical
analysis.
4. Discussion
Recently it was found that AAPs formed 11% of the
marine microbial community in oligotrophic waters of
North East Pacific [3]. The observed ratios of anoxygenic to oxygenic phototrophs (phytoplankton), as reflected in the BChl a/Chl a ratios varied. Early
359
observations in warm oligotrophic waters of the Eastern
Pacific Ocean indicated BChl a/Chl a ratios from 0.7%
up to 10% [2]. Later measurements performed in the
Northeastern Pacific Ocean yielded the ratio of 0.8%
[3], whereas at the station ALOHA (Hawaii, Dec
2002) we obtained ratios of about 2% (Koblı́z̆ek and
Kolber unpublished). Goericke [25] recently reported ratios of 0.1–2% in waters off Southern California. Similarly, our data from the Black Sea showed the ratio
ranging from 0.3% to 2.2% in June 2001 [12]. Somehow
lower numbers were determined in this study ranging
from 0.12% to 0.65%. From the knowledge of BChl a
concentration (8–50 ng L1) and the earlier determined
cellular pigment contents it is possible to estimate AAPs
cell numbers. Using the cellular pigment content of
1.1 · 1016 g BChl a per cell [3] we estimate that, during
the studied period, AAPs accounted for about 7 · 107 to
5 · 108 cells L1. The reported surface bacterial cell
counts from the Baltic Sea for respective season range
from 2 to 5 · 109 cells L1 [26,27], which indicates that
AAPs might have formed 3–10% of the total bacterial
community.
There were two major factors controlling the BChl a
concentration in the Baltic Sea. The first one was related
to the observed south-to-north gradient (see Fig. 3(b)).
The BChl a content was the lowest in the South and
the highest in the North, which displayed the same trend
as the gradient of colored dissolved organic matter as
estimated from water absorption measurements at 412
nm. This might signalize that AAPs distribution was
controlled by the availability of dissolved organic matter. The second factor governing the abundance of BChl
a was the diel cycle (see Fig. 6). The diel cycle may have
caused also the strong variation in BChl a/Chl a ratios
we have observed in the Black Sea in 2001 [12]. Based
on laboratory experiments, we postulate that the decline
of BChl a during the day reflects the inhibition of BChl a
synthesis by the light, which, in combination with concomitant AAPsÕ mortality, resulted in the pigment decline. Furthermore, the BChl a decline analyzed in
terms of a simple exponential behavior provides information on AAPs mortality rates in their natural
environment.
The validity of the BChl a daytime decline as a measure of AAPs mortality is based on two major assumptions. First, the synthesis of BChl a must be fully
inhibited by light. This requirement is satisfied in the
upper photic layer under sufficient irradiance. Under
low irradiance conditions, or in the presence of deep
water mixing, the synthesis of BChl a might not be fully
stopped during the day, and the analysis of BChl a
decay data would underestimate the true mortality rates.
This may explain the observation from 27 August 2003,
when no BChl a decline was observed. During that day a
storm (wind speeds of 15–20 m/s from 12:00 to 16:00,
wave height 1.5 m and partially cloudy) caused a
360
M. Koblı́z̆ek et al. / FEMS Microbiology Ecology 51 (2005) 353–361
strong mixing in the water column down to 15 m. In
such a situation the bacteria might have been mixed below the euphotic zone, which resulted in an incomplete
inhibition of BChl a synthesis. The second assumption
is that the observed loss of BChl a can be attributed
exclusively to AAPs mortality, and is not affected by
other processes such as pigment degradation or bleaching. The high photostability of bacterial reaction center
supports this notion. It was reported that Rhodobacter
reaction centers were stable even at 10,000 lmol photons m2 s1 [21]. In our chemostat experiments an almost ideal wash-out kinetics of BChl a were observed
which proved that no significant BChl a degradation occurred in the cells. Similar results were reported also by
Yurkov and van Gemerden [24]. Although some cellular
BChl a degradation caused by various environmental
stresses cannot be completely ruled out in relatively
slowly growing populations, we assume that the cellular
BChl a degradation does not significantly affect the
BChl a kinetics in rapidly turning over populations as
those observed in the Baltic.
Apart from these two major assumptions there are
two more limitations of the suggested approach. First,
the method requires stable water column conditions to
assure that the same microbial population is sampled
over the diel time-span of the measurement. This
requirement cannot be satisfied under conditions with
strong currents, tides or large changes in the water column mixing. The second assumption is that the mortality determined during the day-light hours equals the
mortality over the entire day. Grazing by protozoa
and death due to the viral infection are usually assumed
to be two major causes of bacterial mortality [28]. If, for
instance, the grazing pressure would be higher during
the daylight hours than during the night, then BChl a
daytime decline would overestimate the true rates averaged for a whole day. Bettarel et al. [29] showed that the
mortality of heterotrophs in the Mediterranean Sea displayed strong diurnal changes. The estimated mortality
rates were the highest before dawn and the lowest before
sunset. If such a strong variation is present, then the
analysis of the BChl a data will only provide information on the mortality rates during the measured period
(i.e. day-light hours). Yet, in spite of all those limitations, the careful analyses of BChl a patterns could
potentially offer a simple, truly in situ approach toward
assessing the dynamics of AAPs communities without
the need of laborious sample preparation and potential
bottle effects.
So far there is no data on AAPs mortality in their
natural environment. For this reason a comparison
can only be made with published data determined for
the entire bacterioplankton. Mortality rates of AAPs
determined in our study (0.7–2 day1) are similar to
bacterial mortality rates (1–2 day1) reported from Californian coastal waters [28]. The available data from
the Northern Baltic (Bothnian Sea, June, 12–15 C)
indicated bacterial mortality rates of about 0.5–0.6
day1 attributable to protist grazing [30]. Viral infection induced mortality was not quantified in this study,
however, available estimates for the virus contribution
to bacterial mortality range from 10% to 50% [26,31].
This translates into total mortality between 0.6 and 1
day1, which corresponds roughly to our estimates
from the Gulf of Gdańsk and Eastern Gotland Basin.
Interestingly, Kolber et al. [2] observed weakly decaying BChl a signals in the tropical Pacific. Analysis of
those data indicates that the AAPs mortality in the
studied region (9 N 104 W) was rather low, about
0.2–0.3 day1 (s1/2 3 days). This is consistent with
estimates of bacterial growth rates 0.1–0.25 day1 reported in a similar region (0 N 140 W) [32,33]. All
these data signalize that the proposed approach might
provide a reasonably accurate estimate of AAPs mortality rates in the marine environment. In conclusion,
the strong BChl a variation observed in this study suggests that, there was a rather rapid turnover of the Baltic photoheterotrophic community (0.7–2 day1) in late
summer 2003.
Acknowledgements
We thank the captain and the crew of the R/V Oceania and the chief scientist Ryszard Hapter for organizing
the cruise. This research was supported by Czech projects GACR 206/03/P079, MSMT LN00A141, the Inst.
research concept AV0Z5020903, and NSF OCE0331449. MKÕs stay aboard Oceania was supported by
the EC 5th Framework Program project CeSSS, no.
EVK3-CT-2002-80004. MK also thanks Dr. Ondr̆ej Prás̆il for the kind accommodation of the avalanche photodiode module. Support from Photon Systems
Instruments Ltd. is also gratefully acknowledged.
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